The present invention relates to Gemini-virus based constructs capable of symptom less, systemic spread in plant host.
Genetic engineering is slowly replacing classical genetic techniques in generation of plants which are resistant to disease, drought, pests or are simply commercially improved.
Genes that provide resistance against biotic as well as abiotic stresses have been successfully introduced into crop plants [see for example, saline soil resistant tomatoes in Zhang, H-X. and Blumwald, E. Nature Biotechnology 19(8):765-768. (2001); potato virus X (PVX) resistant potatoes expressing the viral coat protein in U.S. Pat. No. 5,773,701; fungal resistance in U.S. Pat. No. 6,288,303; insect resistance in plants expressing the Bacillus thuringiensis toxin in Moellenbeck, D. J., et al., Nature Biotechnology 19:668-672 (2001); herbicide resistance in corn in U.S. Pat. No. 7,002,064 7002056 and cabbage]. In another example, the nutritional quality of an important crop such as rice was improved by introducing transgenes that enabled plants to manufacture beta-carotene (a vitamin A precursor) in their endosperm, thus solving vitamin A deficiency in rice eating populations [Ye, X., Science 287:303-305 (2000)]. Indeed, more than 50 genetically modified plants have already been approved by the FDA [Bren, L., FDA Consumer Magazine online issue 37 (2003)].
The latest trend in genetic engineering of crop plants is pharma-crops that produce proteins or chemicals for pharmaceutical and industrial uses. Plants have many advantages as a productive economical source of biomass. Plants lack contamination with animal pathogens, their genetic manipulation is relatively easy, they possess eukaryotic protein modification machinery and therefore are a better industrial protein source than prokaryote or cell line systems. Plants have been used, for example, for the production of human serum albumin [Sijmons, P C, et al., Biotechnology (NY) 8(3):217-21 (1990)], of protein antigens to be used as vaccines [Haq, T A et al., Science.; 268(5211):714-6 (1995)] and for the production of humanized antibodies [Tavladoraki, P., et al., Nature 366, 469-472 (1993)].
Present techniques for DNA delivery into plants include direct as well as indirect methods. However, each of these delivery methods is not without limitations. The direct DNA delivery systems [particle bombardment: Klein, T M et al., Nature, 327, 70-73 (1987); silicon carbide whisker technology (SIC-Kaepplar, H. F., et al., Plant Cell Reports 8: 415-418 (1990); electroporation (D'Halluin et al., 1992)] tend to result in integration of multiple copies of transgenes and are considered to be limited, unpredictable and transient. Indirect approaches [e.g. Agrobacterium: Travella S, Plant Cell Rep. 23(12): 780-9 (2005)] oftentimes result in integration of multiple copies of the foreign DNA into the plant genome along with unwanted sequences from the vector ‘backbone’ [Lange M, et al., Plant Cell Rep. (2006)].
Integration of foreign DNA into the plant genome to become a heritable trait raises many risks. Traits beneficial to crops may, through horizontal gene transfer or hybridization through breeding with wild relatives, provide wild plants with unwanted competitive advantages [(Ellstrand, N. C., et al., Annual Review of Ecology and Systematics 30: 539-63 (1999)]. Also, Transformation with Agrobacterium is a complex process which requires elimination of false positives arising from the growth of Agrobacterium in host tissues, and selection of transformed plants. The use of antibiotic resistance as a marker in the development of transgenic crops has also raised concerns regarding the increase of antibiotic resistance in the environment through horizontal transfer of antibiotic resistance genes to soil micro-organisms. Scientists now have the means to remove marker genes before a crop plant is developed for commercial use [e.g., Iamtham, S., and A. Day, Nature Biotechnology 18:1172-1176 (2000)], but these means involve further costs and tedious procedures. In addition, several species or varieties of plants are still difficult to transform.
Infection of plants with modified viruses is simpler and quicker than the regeneration of stably transformed plants, since plant viruses are small and easy to manipulate, have the inherent ability to enter the plant cell, and will multiply to produce a high copy number of the gene of interest. Viral vectors have been engineered for delivery of genetic material and expression of recombinant proteins in plants [e.g., Pogue, G. P., Annu. Rev. Phytopathol. 40: 45-74 (2002); Gleba, Y., et al., Curr. Opin. Plant Biol. 7: 182-188 (2004); U.S. Pat. Nos. 5,316,931 and 5,811,653 for RNA virus vectors]. Viral expression systems are considered transient expression systems since the viral vectors are not integrated into the genome of the host. However, viral vectors still hold many limitations. Plant viral vectors have the potential to cause disease in their plant hosts, they posses the ability to naturally spread between plants in the field, and in some cases, can be spread through pollen or seed to the next generation. Viral vectors are also limited in their systemic spread in the plant, in host ranges, expression stability, and in the size of insert which can be tolerated [Shepherd, R. J., The Biochemistry of Plants. Ed. A. Marcus, 15, 536-616. Academic Press, New York (1989); Dawson, W. O. et al., Virology 172:285-292 (1989); Covey, S. N. & Hull, R. in Genetic Engineering with Plant Viruses, pp. 217-249, CRC Press (1992); Viaplana et al., 82, 59-65 Journal of General Virology (2001)]. Finally, like transgenic plants, modified viruses are classified as a Genetically Modified Organism (GMO) and thus are subject to regulatory and moral constraints.
Geminiviruses are viruses that possess either one or two single-stranded DNA molecules, encapsidated in twinned “geminate” icosahedral particles. The Geminivirus replicative cycle relies entirely on DNA intermediates and occurs within the nucleus of the infected cell through two basic stages: conversion of ssDNA to dsDNA intermediates and rolling-circle replication, leading to the production of, progeny virus. In Geminiviruses, expression of viral proteins occurs from the transcriptionally active circular dsDNA forms [Gutierrez, C., et al., Veterinary Microbiology 98: 111-119 (2004)].
An example of a Geminivirus is TYLCV, which is a mono-partite begomovirus [Stanely, J. et al., Advances in virus research 30, 139-177, (1985)] with a known genome organization [Hanley-Bowdoin, L., et al., Critical Reviews in Biochemistry and Molecular Biology 35, 105-140 (2000)]. TYLCV infection of tomato presents a serious agricultural-economical problem. TYLCV can not be mechanically inoculated and is transmitted by Bemisia tabaci, but agroinoculation of Geminivirus DNA as an entity longer-than-one-genome-length causes systemic infection [Czosnek, H., et al., Plant Mol. Biol. 22, 995-1005 (1993)].
Until recently insertions into the DNA genome of Geminiviruses for gene expression was successful only if the modified vector is of a size comparable to that of the wild type viral DNA. In monopartite geminiviruses, removal of any viral gene in order to maintain such a size abolished the viral vector's ability to spread systemically [Stanley, J., Curr. Opin. Genet. Dev. 3, 91-96 (1993)]. Introduction of bacterial compatible origin of replication and a multiple cloning site enabled plant expression from a Gemini vector, but the insertion abolished systemic spread, and thus the use of such monopartite Gemini-based expression vectors was confined to cell cultures and endosperm [Ugaki, M. et al., Nucleic Acids Research 19, 371-377 (1991); Tamilselvi. D., et al., Plant Cell Reports 23, 81-90 (2004)], where systemic infection was not required.
Pyrrolnitrin (PRN) is an antifungal and antibacterial compound produced by certain strains of the bacteria Pseudomonas fluorescence and other bacteria such as Burkholderia cepacia (for example, Chernin et al. (1996) Current Microbiology 32:208-212 and El-Banna and Winkelmann (1998) J. Applied Microbiology 85:69-78). The metabolic pathway of PRN production and the functional dissection of its component have been elucidated (for example, Kirner et al. (1998) J. Bacteriol. 180:1939-1943). PRN-producing microorganisms are potential agents for biological control of plants diseases by colonizing the soil with PRN-producing bacteria (for example, Hwang et al. (2002) Biological Control 25:56-63 and Haas and Keel (2003) Annual review of Phytopatology 41:117-153). PRN spraying in field tests reduced disease incidence caused reduction in infectivity of several fungi up to 8-fold. In addition, bacterial genes involved in the PRN pathway were introduced into plants (each was introduced separately), and the resultant transgenic plants carrying 3 transgenes (out of the 4 genes in the operon) were field-tested, reducing disease incident caused by several fungi 3-5-fold. Data on field tests (spraying and transgenic) are documented in U.S. Pat. No. 5,698,425).
There is thus a widely recognized need for, and it would be highly advantageous to have, a transient expression vector devoid of the above limitations.